RNA interference (RNAi) is a phenomenon in which small double-stranded RNA molecules induce sequence-specific degradation of homologous single-stranded RNA. RNAi has been used as a tool to degrade mRNA in cells to shut down the effect of specific genes in many cell-types. In this approach to control gene expression, double-stranded RNAs that are complimentary to known mRNA's are introduced into a cell to specifically target and destroy that particular mRNA. Once double stranded RNA (dsRNA) enters the cell, it is cleaved by a ribonuclease enzyme, dicer, into double stranded small interfering RNAs (siRNAs). The siRNAs become integrated into a multi-subunit protein complex which guides the siRNAs to the target RNA sequence.
In plants, RNAi can be induced through microinjection of long double-stranded RNA or by introduction of DNA constructs that may be transcribed into such double-stranded RNA molecules. The double-stranded RNA is cleaved into RNA fragments of about 19 to 23 nucleotides called interfering RNAs (siRNAs). siRNAs are incorporated into a ribonuclease enzyme complex known as the RNA-induced silencing complex (RISC). The antisense strand of siRNA within the RISC pathway serves as a guide for sequence-specific degradation of homologous messenger RNAs.
The ability of transfected synthetic small interfering RNAs to suppress the expression of specific transcripts has proven to be a useful tool to study gene function. Recently short hairpin RNAs (shRNAs) have been shown to result in gene silencing as effectively as short dsRNAs. Several DNA-based vectors have been developed that direct transcription of small hairpin RNAs (shRNAs). These RNAs are processed into functional siRNAs by cellular enzymes. RNAi vectors for the expression of shRNAs are available. These vectors typically use RNA polymerase III (Pol III) to express short hairpin RNAs. These transcripts adopt stem-loop structures that are processed into siRNAs by the RNAi machinery.
Other vectors have been developed that drive expression of both the sense and antisense strands of a DNA construct separately. The transcripts hybridize in vivo to make the siRNA. In efforts to induce long-term gene silencing, expression vectors that continually express siRNAs in stably transfected cells have been used.
Presently, silencing genes in more than one tissue requires the use of two separate expression cassettes. This approach takes up valuable space in a molecular stack, increasing the regulatory burden for sequence and expression confirmation, as well as, the potential for undesirable rearrangements and deletions. Currently available promoters, particularly tissue-preferred promoters, may not cover the entire temporal range of expression need to achieve the trait goals. Methods and compositions are needed that avoid these complications and allow for differential expression or the increased time-frame of expression of a silencing element without an increase in the size of the expression cassette.